Genetic reporter systems have contributed greatly to the study of eukaryotic gene expression and regulation. Although reporter genes have played a significant role in numerous applications, both in vitro and in vivo, they are most frequently used as indicators of transcriptional activity in cells.1 
Typically, a reporter gene is joined to a promoter sequence in an expression vector that is transferred into cells. Following transfer, the cells are assayed for the presence of the reporter by directly measuring the reporter protein itself or the enzymatic activity of the reporter protein. An ideal reporter gene is one that is not endogenously expressed in the cell of interest and it is amenable to assays that are sensitive, quantitative, rapid, easy, reproducible and safe.
Fundamentally, an assay is a means for translating a biomolecular effect into an observable parameter. While there are theoretically many strategies by which this can be achieved, in practice the reporter assays capable of delivering the speed, accuracy and sensitivity necessary for effective screening are based on photon production.
Photon production is realized primarily through fluorescence and chemiluminescence. Both processes yield photons as a consequence of energy transitions from excited-state molecular orbitals to lower energy orbitals. However, they differ in how the excited-state orbitals are created. In chemiluminescence, the excited states are the product of exothermic chemical reactions, whereas in fluorescence the excited states are created by absorption of light.
Assays based on fluorescence or chemiluminescence can yield high sample throughput. The introduction of the Fluorometric Imaging Plate Reader (FLIPR™, Molecular Devices),2 an image-based instrument capable of measuring 384 wells simultaneously, has allowed extensive use of fluorescent dye-based Ca2+ mobilization assays in the high throughput screening (HTS) process.
New capabilities in chemiluminescence, particularly in bioluminescence (when a chemiluminescent reaction occurs in an organism with the aid of an enzyme), are now adding new bioluminescence techniques to HTS.3 
Bioluminescence is the generation of light by a biochemical reaction involving oxidation of a chemiluminescent substrate (luciferin) via an enzyme (luciferase). Bioluminescence differs from species to species, but the general mechanism begins with the oxidation of a luciferin by its luciferase enzyme in the presence of O2 to form an excited state of the oxidation product (oxyluciferin).4 
Bioluminescence colors can range from blue to red (FIG. 1). The particular wavelength that is emitted depends in part on the type of luciferins employed, and these cover diverse structural classes (FIG. 1, structure 1-4).5 Luciferases from organisms that yield very bright bioluminescence have been adapted for the use as reporters in HTS assays, the most common being from the jellyfish Aequorea Victoria (Aequorin), the sea pansy Renilla reniformis (RLuc), and the firefly Photinus pyralis. Because RLuc uses the same substrate (coelenterazine, 1) as Aequorin, it yields the same products, emits a photon with similar spectral characteristics and the reaction mechanisms are expected to be similar.6 But Aequorin can also function as a calcium dependent luciferase (RLuc itself is not calcium dependent).
This phenomenon has been extensively used in different formats for life science research and drug discovery owing to its extremely high sensitivity, replacing advantageously hazardous methods using, for instance, radioelement.
There are five main problem with radioisotopes,1 placing some practical limitations on their application in the biological laboratory:                1) Preparation of the isotope; this requires a special, costly apparatus such as a nuclear reactor or particle accelerator together with the safety facilities for handling highly radioactive compounds.        2) Hazard; radioactive isotopes are dangerous, not only at the high levels involved in their preparation but also even at the levels in reagents used in research and clinical laboratories. Safety precautions, including special “hot” rooms and cabinets, together with personnel screening are therefore essential. Special facilities are also required for disposal of radioactive waste.        3) Sensitivity and detection speed; the detection limit for a radioisotope can be estimated from the half-time (t1/2) of its decay. The shorter the half life the lower the detection limit: At=A0 e−kt k=0.693/t1/2                     where:                            At=Amount of pure isotope at time t                A0=Amount of pure isotope at time 0                k=decay rate constant=0.693/t1/2                                                 4) The consequences of isotope decay; The most sensitively detectable isotopes by their nature decay rapidly. Some decay is so fast as to make the isotopes practically useless. A further problem is the effect of decay on the molecule to which the isotope is attached. Biological molecules, such as proteins, are particularly susceptible to radiolytic damage.        5) The need for a separation step; virtually all analytical applications of radioisotopes require a separation step in order to isolate the appropriate material for radioactive counting. These separation steps introduce imprecision, are sometimes laborious, and complicate automation for clinical application.        
Chemiluminescent labels are groups of synthetic organic compounds (e.g. luminol, acridinium esters), cofactors in bioluminescent reactions (ATP, NAD), enzymes (peroxidase, oxidase, kinases, luciferases), and represent a real alternative to a radioimmunoassay and to develop new approaches to advance our knowledge of how cells work.1 
A large number of luminescent labels can be readily detected within a few second in the fmol-amol range (10−15-10−18).1 The improvement of peroxydase “enhancers”, the use of luciferins with a higher quantum yield and lower chemical “noise” than luminol,1 and the use of better peroxidase (e.g. luciferase peroxydase), may enable tipomol (10−21) sensitivity to be achieved.
Image intensifiers are available for detecting chemiluminescence as sensitively as a photomultiplier tube,7 beyond the intensity per unit area necessary to produce an image in the naked eye. Chemiluminescent labels are therefore applicable to light microscopy. A label producing a continuous glow, e.g. peroxydase or luciferase, may be more convenient than one producing a flash.
Several types of homogeneous assay, not requiring a separation step, have been established using chemiluminescent labels. The labelled reagents have been found to have a shelf life of many years. Many clinically useful assay have been established which satisfy the normal criteria for a good immunoassay.1 The working range can be established by evaluation of the precision profile and good correlation (r>0.95) with its checked radioactive counterpart. The chemiluminescent labels are safe to handle and, unlike radioisotopes, no special precautions appear necessary. Many luciferins are now commercially available and they're relatively easy to synthesise in gram quantities.
Historically,1 changes in intracellular Ca2+ levels have been readily detected using fluorescent dyes that emit light in proportion to the changes in intracellular Ca2+ concentration. An alternative approach to indirectly measure changes in Ca2+ concentrations involves the use of recombinantly expressed biosensor photoproteins, of which Aequorin is a prototypic example (see Table 1).8
TABLE 1Comparison of Ca2+-sensitive dyes and photoproteins for HTS.PropertiesCa2+-sensitive dyesaPhotoproteinsbGFP sensorscEnergy emissionFluorescenceChemiluminescenceFluorescenceDetection deviceFLIPRFLIPR, CCDMicroscopyBackgrouns Ca2+Moderate to highLowModerate/highSignal-to-noise ratioLow to moderateHighModerateDynamic range of Ca2+ changeLow to moderateHighHighSensitivityLow to moderateHighHighAdaptability to assay conditionsLowModerate/highLowCells per well neededThousandsHundredsSingle/hundredsEase of use in HTSHighHighModerateCompount interferenceModerateNoneModerateUseful for orphan GPCRsNot easyYesModerateTargeted to subcellular sitesNoYesYesCa2+ in microdomainsNoYesYesReaction kineticsFastPhotina slowFastUsed in vivoNoYes but rarelyYes
The bivalent ion Ca2+ is a critical second messenger involved in many physiological and signal transduction processes within a cell.9 The central role of Ca2+ in intracellular signalling, and its physical/chemical properties also makes it an attractive reporting molecule for drug discovery (for example, GPCRs, ion channels, and ion exchangers are among the most interesting target classes for the pharmaceutical industry).8 The use of photoproteins as indicators of Ca2+ activation in HTS offers many advantages: low background levels result in a large signal to noise ratio, Ca2+ concentrations can be measured at specific cellular sites, tested compounds only require short incubation periods, and reaction kinetics can be followed.
Aequorin, has been validated for functional assays of many GPCRs, and the results obtained are comparable to those obtained with the use of fluorescent dyes. However, the adaptation of Aequorin assays to HTS is not always easy due to the low quantum yield of aequorin and the fast kinetics of the reaction.
Coelenterazines are small and hydrophobic molecules that readily cross cell membranes, thereby permitting analysis of intact cell. The donor emission spectrum dependent upon the coelenterazine used, in addiction to the donor protein itself.
The luminescence reaction catalyzed by Renilla luciferase (RLuc) is shown in Scheme 1. RLuc catalyzes the enzymatic degradation of coelenterazine, leaving the resultant product, coelenteramide 5, with an electron in an excited state.10

It has been suggested that the blue light emission (λ=481 nm peak for RLuc) associated with coelenterazine bioluminescence is due to the excited state coelenteramide existing in its amide anion form when it is in the protein's enzymatic pocket.11 
However, at least in the case of photoproteins that use coelenterazine such as Obelin, Aequorin, more recent literature has favored assigning the phenolate anion as the blue emitting species in bioluminescence.12 
Since the 1970s, a large number of coelenterazine analogs were synthesized to obtain new molecules with improved properties.13,10 In particular, red shifted emission peak, high quantum yield and better sensibility of Ca2− ions are still needed.
Examples of bioluminescence applications that use the Apoaequorin/Coelenterazine system, include: reporter assays, measuring Ca2− in cells, reactive oxygen species (ROS) detection, protein interaction studies.
Concerning reporter assays, aequorin has been largely used to tag (by plasmid or other engineering methods), recombinant proteins, and then monitor their expression (localization, regulation . . . ).14 For imaging bioluminescent reporters15 in intact animals it is highly advantageous for the luciferase to emit a large percentage of its photons in red to near-infrared wavelengths (600-900 nm), as tissue attenuation of optical wavelength photons is minimized in this region of the spectrum1. The luciferases that use coelenterazine as their substrate (for example, RLuc, Gaussia princeps luciferase) have served as useful adjuncts to the beetle luciferases in both cell culture and animal experiments.16 They also have been used in situations where the ATP-dependence and poor thermal stability of the beetle luciferases would be a liability, such as in bioluminescently tagged imaging probes17 and self-illuminating quantum dots.18 
However these luciferases have a major limitation for most small-animal imaging applications in that their spectral peaks are in the blue region of the visible spectrum (RLuc, λ=481 nm). From locations deeper than subcutaneous depths, most of the photons that make it out of the animal being imaged are few red-shifted wavelength photons.19 
However, the use of native coelenterazine in small animal imaging has been hampered by its λ=481 nm peaked emission spectrum as blue wavelengths, that are strongly attenuated in biological tissues.
To overcome this difficulty, research is focused on red-shifting RLuc through a combination of semirational and random mutagenesis, yielding variants with bathochromic shifts of up to 66 nm. Analysis of the most promising variant demonstrated that it was 6-fold brighter than RLuc, and its green emission spectrum (λ=535 nm peak) generated a further 3-fold improvement in photon transport at depths of 1-2 mm of animal tissuc.19 
The interaction between the luciferase and coelenterazine is not well understood, a conclusive answer cannot be given at this time, but it may be that the pyrazine anion of coelenteramide represents a limit to the bathochromic shift that this luciferin-luciferase system can accomplish. Further, redshifts will most likely involve altering the structure of the luciferin, as shown with coelenterazine-v.10 This particular analog, however, suffers from issues of stability that limits its applicability. Thus, the development of alternative red-shifted coelenterazine analogs more appropriate for small-animal imaging are needed. Another negative aspect of the blue emission of coelenterazine analogs has to be considered. Indeed, due to the high energy of their luminescent intermediates, these molecules show a flash emission which makes the applicability to energy transfer assays difficult.
Consequently, a very fast kinetic of the bioluminescent reaction (few seconds) is observed. The problem is particularly evident in BRET (Bioluminescence Resonance Energy Transfer) where the real time monitoring of biological events needs luminescent substrate with prolonged decay kinetics.20 To avoid this difficulty, recently modified analogs EnduRen™, ViviRen™ (Promega) were synthesized. EnduRen™ and ViviRen™ (in vivo Renilla Luciferase substrate) are a uniquely engineered coelenterazine-based compounds with protected oxidation sites (see Scheme 2).

These modifications are designed to minimize substrate degradation and autoluminescence. It is reported that these substrates may have a longer kinetic output when compared to the native coelenterazine substrates when used in an in viva imaging application in a mouse model. Once inside the cell, the protective groups of the substrate are cleaved by intracellular esterases, generating coelenterazine which reacts with RLuc to produce light. Peak luminescence of EnduRen™ is achieved after 1.5 hours of substrate addition to cells, and signal is stable for >24 hours. Peak luminescence of ViviRen™ is achieved after 2 minutes of substrate addition to cells, with signal half-life from 8-15 minutes.
While a wide number of variants of luciferase, obtained through a combination of semi-rational and random mutagenesis, show bathochromic (red-shifted) shifts, the finding of new coelenterazine analogs with improved properties is lacking.
e-Coelenterazine seems to be the more interesting molecule with a red-shifted signal (λ=510 nm maximum peak of bioluminescence), good quantum yield and high sensibility to Ca2+ concentrations. But this molecule presents difficulties for its synthesis and poor stability.13a 
Literature reports a large number of analogs of the native coelenterazine,13 with a red-shift of the emission peak of chemiluminescence reaction. Among compounds that have shown the best chemiluminescent properties, many of them bear structural modifications which do not allow the correct fitting of the molecules in the enzymatic binding pocket, and, as expected, no bioluminescence signal was detected. Another problem with the modifications proposed was the reduced lipophilicity of molecules and consequent poor cellular membranes crossing. These compounds were synthesized to better understand the structural properties of the molecules related to the chemiluminescent mechanism. The experiments performed show interesting results;11 for example, chemiluminescence efficiency, or quantum yield, is lower than bioluminescence's one. The disparity between the chemiluminescent and bioluminescent efficiencies of coelenterazine is attributed to both the conformational stability of the emitter in the protein and the hydrophobic environment surrounding the coelenteramide anion. To test the conformational stability effects on the chemiluminescence of coelenterazine, analogs with rigidifying bridges were synthesized (6-10; Table 2).13e
TABLE 2 CompoundsR1R2R3R4ΦCl (Relative Light Yield)1HHHOH0.21% (1.0) 6—CH2—HOH0.31% (1.5) 7—CH2CH2—HOH0.48% (2.3) 8—(CH2)3—HOH0.31% (1.5) 9—CH2CH2OHHHOH0.10% (0.46)10—CH2CH2—OHOH  0% (0.01)
Chemiluminescence experiments using compounds with a rigid p-hydroxyphenyl group showed increased ΦCI, with the six-membered ring derivative 7 having the highest quantum yield.
Intramolecular hydrogen bonding effects between the hydroxyl and nitrogen atoms on the imidazopyrazine core decreased the chemiluminescent efficiencies (9,10), supporting the notion that the bioluminescence reaction takes place in a hydrophobic environment in the luciferase enzyme.13e Coelenterazine analogs have also been modified to produce bimodal chemiluminescent systems. Typically, imidazopyrazinones can emit blue to yellow light from the excited singlet state of the amide anion in both acidic and basic environments.21 For other examples of bioluminescence applications, the regeneration of an active semi-synthetic Aequorin, from Apoaequorin produced in cells and a coelenterazine analogue, is a key step in measuring Ca2− in cells. The relative rates of the regeneration of semi-synthetic Aequorins from Apoaequorin and synthetic coelenterazine analogs were compared13a. Another application is the detection of superoxide and peroxynitrite anion (ROS)22. ELISA, bioluminescence resonance energy transfer (BRET) can be used for protein interaction studies.23,20 
In the original BRET technique, oxidation of coelenterazine 1 results in a λ=475 nm Rluc emission peak. Use of the coelenterazine derivative DeepblueC™ (also known as coelenterazine-400a, di-dehydro coelenterazine) results in a λ=395 nm Rluc emission peak.
Due to rapid development in molecular biological tools and recent development of extremely sensitive photon detectors,24,7 Fluorescent imaging (FLI) and Bioluminescent imaging (BLI) can be applied to study cell and tissue specific promoters, but also to follow trafficking and fate of GFP and/or Luciferase expressing cells, apoptosis, protein-protein interaction and gene-transfer.25 
The bioluminescent and chemiluminescent mechanisms are similar for coelenterazine but the conditions for eliciting each reaction are different. In bioluminescence, coelenterazine is the substrate for the photoprotein Aequorin and produces blue light in the presence of Ca2+. On the other hand, chemiluminescent reactions of coelenterazine are typically performed in a purely chemical system with O2 dissolved in an aprotic polar solvents such as dimethylsulfoxide (DMSO), dimethylformamide (DMF), and hexamethylphosphoramide (HMPA) in the presence or absence of bases (e.g., NaOH, t-BuOH, t-BuOK) or acetate buffer.26 
Synthetic chemistry has played a key role in studying the luminescence properties of coelenterazine derivatives. The two synthetic approaches used to obtain 1 and its analogs are the “classical” synthesis13d and a more modern method which utilizes palladium coupling.27,28,29,21 Further improved procedure was performed for an industrial approach.30,31,32 
The present invention aims to solve several negative properties of the coelenterazine analogs present in the market. In particular, the structural modification proposed can be incorporated (easy variation in the synthesis procedure) into the known structures and selected to be the preferred compounds for both basic and industrial research (see Table 3).
TABLE 3Known coelenterazine analogs present in the market λ max.RelativeHalf-riseEmissionLuminescenceRelativeTime (s) MWR1R2R3(nm)capacityIntensity(ms)Coelenterazine Native423.50OHOHPhe4651.001.000.4-0.86-30 msCoelenterazine cp415.48OHOHCP4420.95200.15-0.3 0.63285 msCoelenterazine e449.50OHOHPhe405 and 4650.540.15-0.3 Coelenterazine f425.45FOHPhe 4730.80180.4-0.80.80206-30 msCoelenterazine fcp417.48FOHCP4520.571350.4-0.8Coelenterazine h407.50HOHPhe4640.8210 0.4-0.800.75166-30 msCoelenterazine hcp399.49HOHCP4440.671900.15-0.3 0.655002-5 msCoelenterazine i533.36 IOHPhe4760.700.038Coelenterazine ip389.45IOH2P4410.54471Coelenterazine n457.52NaphOHPhe4670.260.0150.250.156-30 msCoelenterazine 2-methyl331.37N/AN/AN/AN/ACoelenterazine 400a391.46400N/AN/AN/AHydrogen (H), hydroxyl (OH), Phenyl (Phe), CycloPentyl (CP), 2-propionyl (2P), Naphthyl (Naph), methyl (Met). Coelenterazine-e has a —CH2CH— bridge between the 6-phenyl-OH and position 5 of the imidazopyrazinone core.
The compounds of the invention are endowed with an improved red-shifted photoemission in bioluminescence while maintaining lipophilicity and enzymatic binding properties of the original coelenterazine molecules. Yet, the efficiency and sensibility to Ca2− depends on the interactions of the particular compound in the enzymatic binding pocket and the environment involved. Indeed, the bivalent sulfur and selenium atoms are bioisosteres of the methylene group. The present invention relates to new photochemical entity obtained by modification of the methylene (—CH2—) in position C-8 of the imidazo[1,2-a]pyrazin-3(7H)-one nucleus, while the other substituents in position C-2, C-5 and C-6 are preserved (Formula 1).